Evolution of variable lymphocyte receptor B antibody loci in jawless vertebrates

Abstract

Three types of variable lymphocyte receptor (VLR) genes, VLRA, VLRB, and VLRC, encode antigen recognition receptors in the extant jawless vertebrates, lampreys and hagfish. The somatically diversified repertoires of these VLRs are generated by serial stepwise copying of leucine-rich repeat (LRR) sequences into an incomplete germline VLR gene. Lymphocytes that express VLRA or VLRC are T cell-like, while VLRB-expressing cells are B cell-like. Here, we analyze the composition of the VLRB locus in different jawless vertebrates to elucidate its configuration and evolutionary modification. The incomplete germline VLRB genes of two hagfish species contain short noncoding intervening sequences, whereas germline VLRB genes in six representative lamprey species have much longer intervening sequences that exhibit notable genomic variation. Genomic clusters of potential LRR cassette donors, fragments of which are copied to complete VLRB gene assembly, are identified in Japanese lamprey and sea lamprey. In the sea lamprey, 428 LRR cassettes are located in five clusters spread over a total of 1.7 Mbp of chromosomal DNA. Preferential usage of the different donor cassettes for VLRB assemblage is characterized in our analysis, which reveals evolutionary modifications of the lamprey VLRB genes, elucidates the organization of the complex VLRB locus, and provides a comprehensive catalog of donor VLRB cassettes in sea lamprey and Japanese lamprey.

Keywords: adaptive immunity; immune system evolution; jawless vertebrates; transposable elements; variable lymphocyte receptor.

Fig. 1.

Comparative analysis of incomplete germline VLRB genes in two hagfish and six lamprey species. (A) Genomic configurations of germline VLRB genes. The relatively large VLRB intervening sequences in the lampreys contain multiple TE sequences, the classification of which is indicated by color coding: orange (LTR retrotransposon), yellow (non-LTR retrotransposon), gray (endogenous retrovirus), and green (DNA transposon). Except for the germline VLRB in M. mordax (short-headed lamprey), which possesses a multiplex cassette encoding LRRNT-LRR1-LRRV–N-terminal LRRV, the VLRB genes in other lamprey species all contain a 5′LRRCT cassette. However, L. camtschaticum (Japanese lamprey) has two 5′LRRCT cassettes (shown as A and B) in the intervening region, whereas P. marinus (sea lamprey) and L. reissneri (Far Eastern brook lamprey) have another inv-5′LRRCT cassette (cassette B) in the partial VLRB duplicate and in the complete VLRB duplicate germline gene, respectively. This partial duplicate of VLRB in the sea lamprey is located downstream of the germline gene (see Fig. 4) as is the complete duplicate of VLRB in the Far Eastern brook lamprey. The figure is not drawn to scale. (B) The phylogenetic relationships of the eight representative agnathan species (Cyclostomata) are adapted from refs. , , and . The closer phylogenetic relationship between the two Southern Hemisphere lamprey lineages is suggested but remains to be validated with genome-wide dataset (38). (C) Phylogenetic tree based on germline VLRB N-terminal coding regions. (D) Phylogenetic tree of germline VLRB C-terminal coding regions, and (E) Phylogenetic tree of the intervening region 5′LRRCT cassettes of germline VLRB genes. The VLRB phylogenetic trees (C–E) were constructed by the neighbor-joining method. The scientific names of the organisms are denoted with a two-letter code at the end of each branch: Ga, G. australis; Lc, L. camtschaticum; Lr, L. reissneri; Lp, L. planeri; Mm, M. mordax; Pm, P. marinus. The numbers next to each node indicate bootstrap confidence values after 1,000 replications.

Fig. 2.

Usage of 5′LRRCT donor cassettes located in the intervening sequence of the germline VLRB gene in Japanese lamprey (Lc), European brook lamprey (Lp), and sea lamprey (Pm). The Lc VLRB gene contains two inv-5′LRRCT cassettes (designated as A and B), whereas the Pm VLRB gene has one inv-5′LRRCT cassette (designated as cassette A) and, in addition, a partially duplicated VLRB sequence together with an inv-5′LRRCT cassette (designated as cassette B). The genomic donor cassette sequence is shown in each upper panel and three examples of mature VLRB with their accession numbers are shown immediately below. Nucleotide sequence mismatches between duplicate copies of inv-5′LRRCT cassettes in Japanese lamprey and sea lamprey are highlighted in green. The codons encoding the conserved LRRCT cysteine residues are boxed for orientation.

Fig. 3.

VLRB locus organization in the Japanese lamprey. The incomplete germline VLRB gene (red box in Scaffold00524) and 384 donor cassettes are distributed in 22 genomic scaffolds and 91 contigs (SI Appendix, Table S2). Three representative VLRB scaffolds are shown here because of their length and large number of constituent VLRB donor cassettes. Presumptive duplication events for VLRB-encoding donor cassettes (indicated by background yellow colors) are connected by dotted black line. Frequently used donor cassettes are indicated by an asterisk. Arrowheads above individual donor cassettes indicate their reverse orientation relative to other donor cassettes in a particular scaffold. In scaffold00104, a 5′LRRCT cassette is within the intron of the CHST14 gene. The fragmented genome assembly precludes determination of the relative order and orientation of the scaffold. The illustrated components are not drawn to scale and regions of unresolved sequence are ignored. A cartoon of an assembled VLRB is shown in the black box at the bottom right).

Fig. 4.

Genomic map of the sea lamprey (P. marinus) VLRB locus. (A) VLRB components are shown in same orientation as the VLRB germline gene and in reverse complement to the VLRB encoding genomic reference sequence (NCBI accession no. NC_046089.1). VLRB donor cassettes in three regions of the chromosome are listed in the same orientation as the core germline VLRB gene: cluster I, a region with 167 cassettes spanning from 12.0 to 12.6 Mbp in the NC_046089.1 sequence; cluster II, spanning from 9.3 to 9.9 Mbp in the NC_046089.1 sequence, contains the germline VLRB gene, the partial duplication of the germline VLRB, and 178 donor cassettes; cluster III, a group of 13 5′LRRCT cassettes at 1.5 Mbp in the NC_046089.1 sequence. VLRB cassettes are distributed in the regions indicated with blue shading. The positions corresponding to the segments of the three Japanese lamprey scaffolds diagrammed in Fig. 3 (Lc_Sca00104, Lc_Sca00524, Lc_Sca00150) are shown as lines under the chromosome map corresponding to the three homologous sea lamprey genomic segments. Sequence for the entire locus is defined except for two small unresolved regions of 1,539 bp and 17,802 bp located well outside of the donor cassette clusters. (B) An expanded view of the VLRB regions containing clusters I to III shown in light blue shading with cassettes indicated with colored ovals and associated scale bars. The germline VLRB gene and its partial duplicate (see SP, signal peptide and C-term, C-terminal labels) are in the same transcription orientation in cluster II. A map of the sea lamprey CHST14L gene with a 5′LRRCT cassette encoded in an intron (red rectangle) is shown on the right. An arrow indicates its position flanking the VLRB cluster I, as it does also in L. camtschaticum Sca00104 (Fig. 3). Arrowheads indicate transcriptional orientation. (C) A dot plot of alignments between sea lamprey sequence containing VLRB (NC_046089.1) and the homologous VLRB encoding sequence from the recent Japanese lamprey, L. camtschaticum, assembly (scaffold MU249593.1). Clusters I, II, and III are positioned similarly in genome assemblies from both species. Donor cassettes are restricted to the regions encompassed by the boxes in both species. A duplication and inversion of part of cluster II is evident in the Japanese lamprey (red arrow). An expanded view of cluster III is shown. Matching among different cassettes within clusters is evident in the checkered array of alignment marks within the three boxed cluster regions. Green marks indicate forward matches, red marks indicate reverse complement matches relative to the GenBank sequences.

Fig. 5.

Multiple segments of LRR cassettes may contribute to VLRB assembly in the Japanese lamprey. Two representative multiplex LRRV cassettes are shown: the donor cassette (A) potentially encode three modules (i.e., LRRV-LRRV-LRRV), whereas the one in B encodes six LRRV modules in two different reading frames (a single base pair insertion is indicated in blue). Assembled mature VLRB sequences, numbered and highlighted in different colors, are shown below the genomic donor cassette along with their accession numbers. The hypothetical translation of the putative LRR donor cassette is shown above the cassette sequence.